Cytokines are a diverse group of small proteins that mediate cell signaling/communication. They exert their biological functions through specific receptors expressed on the surface of target cells.
Cytokines can be subdivided into several groups, including the immune/hematopoietins, interferons, tumor necrosis factor (TNF)-related molecules, and the chemokines. Representative immune/hematopoietins include erythropoietin (EPO), granulocyte/macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), leukemia inhibition factor (LIF), oncostatin-M (OSM), ciliary neurotrophic factor (CNTF), growth hormone (GH), prolactin (PRL), interleukin (IL)-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, and IL-12. Representative interferons (IFN) include IFN.alpha., IFN.beta., and IFN-gamma. Representative TNF family members include TNF.alpha., interferon (IFN).beta., gp.sup.39 (CD40-L), CD27-L, CD30-L, and nerve growth factor (NGF). Representative chemokines include platelet factor (PF)4, platelet basic protein (PBP), gro.alpha., MIG, ENA-78, macrophage inflammatory protein (MIP)1.alpha., MIP1.beta., monocyte chemoattractant protein (MCP)-1, I-309, HC14, C10, Regulated on Activation, Normal T-cell Expressed, and Secreted (RANTES), and IL-8.
IFN-gamma
IFN-gamma was first described 30 years ago as an antiviral agent (Wheelock, 1965). Since that time the protein has been shown to be a remarkably pleiotropic cytokine which plays important roles in modulating virtually all phases of immune and inflammatory responses. The cDNAs for murine IFN-gamma (Gray and Goeddel, 1983) and human IFN-gamma (Gray and Goeddel, 1982) have been cloned, sequenced, and characterized.
IFN-gamma is a member of a family of proteins related by their ability to protect cells from viral infection. This family has been divided into three distinct classes based on a variety of criteria, IFN-alpha (originally known as Type I IFN or Leukocyte IFN), IFN-beta (also originally known as Type I IFN or Fibroblast IFN) and IFN-gamma (originally known as Type II IFN or Immune IFN). IFN-gamma is unrelated to the Type I interferons at both the genetic and protein levels (Gray, et al., 1982). The human and murine IFN-gamma proteins display a strict species specificity in their ability to bind to and activate human and murine cells. This is due at least in part to their modest homologies at both the cDNA and amino acid levels (60% and 40% respectively).
IFN-gamma is produced by a unique set of stimuli and only by T lymphocytes and natural killer (NK) cells. The human and murine genes for IFN-gamma are 6 kb in size, and each contain four exons and three introns. These genes have been localized to human chromosome 12 (12q24.1) and murine chromosome 10. Activation of the human gene leads to the transcription of a 1.2 kb mRNA that encodes a 166 amino acid polypeptide (Derynck, et al., 1982). The human protein contains a 23 residue amino terminal hydrophobic signal sequence which gets proteolytically removed, giving rise to a mature 143 residue positively charged polypeptide with a predicted molecular mass of 17 kDa. Variable post-translational enzymatic degradation of the positively charged carboxy terminus (Rinderknecht, et al., 1984) is responsible for the charge heterogeneity of the fully mature molecule. Proteins with six different carboxy termini have been detected for both natural and recombinant forms of IFN-gamma. Two polypeptides self-associate to form a homodimer with an apparent molecular mass of 34 kDa (Scahill, et al., 1983). The homodimer is the biologically active form of the protein. Mature human IFN-gamma contains no cysteine residues, thus the homodimer is held together entirely by noncovalent forces. This quaternary structure of the native protein explains its characteristic sensitivity to extremes of heat (protein denatured at temperatures above 56.degree. C.), and pH (activity rapidly lost at pH values less than 4.0 and greater than 9.0) (Mulkerrin and Wetzel, 1989).
The remarkable pleiotropic effects of IFN-gamma are mediated through binding to a single type of IFN-gamma receptor. The structure and function of murine and human IFN-gamma receptors have been described (Schreiber, et al., 1992). These receptor proteins are expressed on nearly all cells (except erythrocytes), and platelets (Anderson, et al., 1982). The receptor binds ligand with high affinity (Kd=10.sup.-9 -10.sup.-10 M) and is expressed on most cells at modest levels (200-25,000 sites/cell). Upon IFN-gamma binding to the receptor at the cell surface, the intracellular domain of the receptor is phosphorylated at serine and threonine residues (Hershey, et al., 1990).
One of the major physiologic roles of IFN-gamma is as a regulator of immune response induction, specifically its ability to regulate expression of class I and II major histocompatibility (MHC) antigens on a variety of immunologically important cell types (Trinchieri and Perussia, 1985) Functionally, IFN-gamma dependent upregulation of MHC gene expression is an important step in promoting antigen presentation during the inductive phase of immune responses and may play a role in antitumor activity of IFN-gamma (Buchmeier and Schreiber, 1985).
Another major physiologic role for IFN-gamma is its ability to activate human macrophage cytotoxicity (Schreiber and Celada, 1985). Therefore, IFN-gamma is the primary cytokine responsible for inducing nonspecific cell-mediated mechanisms of host defense toward a variety of intracellular and extracellular parasites and neoplastic cells (Bancroft, et al., 1987). This activation is a result of several distinct functions of IFN-gamma. IFN-gamma has been shown to effect the differentiation of immature myeloid precursors into mature monocytes (Adams and Hamilton, 1984). IFN-gamma promotes antigen presentation in macrophages, through the induction of MHC class II expression as described above, but also by increasing levels of several intracellular enzymes important for antigen processing (Allen and Unanue, 1987). Macrophage cell surface proteins such as ICAM-1 are upregulated by IFN-gamma, thus enhancing the functional results of the macrophage-T cell interaction during antigen presentation (Mantovani and Dejana, 1989). IFN-gamma activates the production of macrophage derived cytocidal compounds such as reactive oxygen- and reactive nitrogen-intermediates and tumor necrosis factor-a (TNF-a) (Ding, et al., 1988). IFN-gamma also reduces the susceptibility of macrophage populations to microbial infections (Bancroft, et al., 1989). Animal models have been used to study the role of IFN-gamma in the clearance of microbial pathogens. Neutralizing monoclonal antibodies to IFN-gamma were injected into mice before infecting them with sublethal doses of various microbial pathogens. These mice lost their ability to resolve the infection initiated with Listeria monocytogenes (Buchmeier and Schreiber, 1985), Toxoplasma gondii (Suzuki, et al., 1988), or Leishmania major (Green, et al., 1990).
Besides these nonspecific cell mediated cytocidal activities, IFN-gamma also enhances other macrophage immune response effector functions. IFN-gamma up-regulates expression of Fc receptors on monocytes/macrophages (FcgRI), thus enhancing the capacity of the macrophage for antibody dependent cell killing (Erbe, et al., 1990). IFN-gamma also promotes humoral immunity through enhancement of complement activity. It does this in two ways, i) by promoting the synthesis of a variety of complement proteins (ie., C2, C4, and Factor B) by macrophages and fibroblasts, and ii) by regulating the expression of complement receptors on the mononuclear phagocyte plasma membrane (Strunk, et al., 1985).
IFN-gamma also exerts its effects on other cells of the immune system. It regulates immunoglobulin isotype switching on B cells (Snapper and Paul, 1987). IFN-gamma plays a positive role in the generation of CD8.sup.+ cytolytic T cells (CTLs) (Landolfo, et al., 1985) and enhances NK cell activity. Recently, it has been unequivocally established that CD4.sup.+ T cells do not constitute a homogeneous class of cells. Indeed, a paradigm of lymphokine biology and of the function of CD4+ T cells has arisen, the so-called Th1/Th2 paradigm (for a review see Paul and Seder, 1994). The T.sub.H1 clones, through their production of IFN-gamma, are well suited to induce enhanced microbicidal and antitumor activity in macrophages as detailed above (enhanced cellular immunity), while the Th2 clones make products (IL-4, IL-5, IL-6, IL-10, IL-13) that are well adapted to act in helping B cells develop into antibody-producing cells (enhanced humoral immunity). The role played by IFN-gamma at this crucial nexus of T cell effector function is fundamental to the success or failure of the immune response.
IFN-gamma plays a major role in promoting inflammatory responses both directly, and indirectly through its ability to enhance TNF-.alpha. production. During an inflammatory response, cells leave the circulation and migrate to the point of infection. During this process they must first bind to and then extravasate through vascular endothelium. Both IFN-gamma and TNF-.alpha. can promote the expression of overlapping sets of cell adhesion molecules (ICAM-1, E-selectin, and others) that play an important role in this process (Pober, et al., 1986; Thornhill, et al., 1991). In fact, experiments have shown that these two cytokines exhibit synergistic effects in up-regulating cell adhesion molecules in vivo (Munro, et al., 1989). One can envision microbial infections in which the microorganism is already widespread at the time the immune response develops or in which the response does not quickly rid the host of the infectious agent. This results in continued T cell activation inducing both local inflammation and tissue damage with ensuing loss of normal function. Indeed, when the infectious agent is of little intrinsic pathogenicity, the disease induced by the infection may largely reflect the consequences of such a response.
Excessive production of IFN-gamma may play a role in autoimmune disorders (for review see Paul and Seder, 1994 and Steinman, 1993). The mechanism for this may involve excessive tissue damage due to aberrant or enhanced expression of class I and class II MHC molecules or the role of an excessive T.sub.H1 cellular response. A role for IFN-gamma and the tissue-damaging effects of immune responses mediated by T.sub.H1 -like cells has been suggested in autoimmune disorders such as rheumatoid arthritis (Feldmann, 1989), juvenile diabetes (Rapoport, et al., 1993), myasthenia gravis (Gu, et al., 1995), severe inflammatory bowel disease (Kuhn, et al., 1993), and multiple sclerosis (Traugott, 1988).
IL-4
Interleukin-4 (IL-4) is a remarkably pleiotropic cytokine first identified in 1982 as a B cell growth factor (BCGF) (Howard, et al., 1982). In that same year, IL-4 was identified as an IgG1 enhancing factor (Isakson, et al., 1982). Because of the effect IL-4 has on B cells, it was first called BCGF-1, later termed BSF-1 (B-cell stimulatory factor-1), and in 1986 it was given the name IL-4. The cDNAs for murine IL-4 (Noma, et al., 1986; Lee, et al., 1986) and human IL-4 (Yokota, et al., 1986) have been cloned, sequenced, and characterized.
IL-4 can be regarded as the prototypic member of a family of immune recognition-induced lymphokines designated the "IL-4 family" (for a review see Paul, 1991). This family consists of IL-4, IL-5, IL-3, and granulocyte-macrophage colony-stimulating factor (GM-CSF). The properties shared by these proteins leads to this grouping and include, i) the linkage of the genes for the members of the family (van Leeuwen, et al., 1989), ii) the action of each member of the family as a hematopoietic growth factor in addition to any effects it may exert on lymphoid cells, iii) the receptors for these proteins are all members of the hematopoietin family of receptors (Bazan, 1990a), and iv) coexpression of these factors by a subpopulation of cloned CD4.sup.+ T cells (the so-called T.sub.H2 cells) (Mosmann, et al., 1989) and by mast cells (Plaut, et al., 1989).
The remarkable pleiotropic effects of IL-4 are mediated through binding to cell surface receptors (IL-4R). The murine IL-4R (Mosely, et al., 1989; Harada, et al., 1990), and the human IL-4R (Idzerda, et al., 1990; Galizzi, et al., 1990) have been cloned, sequenced, and characterized. IL-4R are present on a variety of hematopoietic (Park, et al., 1987) and nonhematopoietic cells (Lowenthal, et al., 1988). On both human and murine resting T and B cells, IL-4R are present in low numbers (&lt;400) and are regulated by cytokines and other factors. The receptor binds IL-4 with high affinity (Kd=10.sup.-10 M). Now that most of the receptors for immunoregulatory and hematopoietic cytokines have been cloned, it is apparent that the majority of these receptors fall into a large family. This hematopoietic cytokine receptor superfamily includes receptors for IL-4, IL-2 (.beta. and .gamma. chains), IL-7, IL-9, and IL-13 which modulate the lymphoid system; and receptors for erythropoietin, granulocyte-colony stimulating factor (G-CSF), GM-CSF, IL-3, and IL-5 which modulate the hematopoietic system. The superfamily also includes receptors for factors believed to normally function outside the immune and hematopoietic systems, including receptors for growth hormone (GH), prolactin, leukemia inhibitory factor (LIF), IL-6, IL-11, and ciliary neurotrophic factor (CNF) (for a review see Kishimoto, et al., 1994).
A general first step in the signaling processes of immune and hematopoietic cytokines may be ligand-induced dimerization of receptor components whose cytoplasmic regions interact to initiate a downstream signaling cascade. The IL-4 receptor has a long putative intracellular domain (553 amino acids in mouse, 569 in human) with no known consensus sequences for kinase activity or for nucleotide-binding regions. The biochemical nature of signals induced by the binding of IL-4 to its receptor have not been elucidated. It does appear that the cytosolic domain of the receptor is essential for its signaling function (Mosely, et al., 1989). Ligand induced dimerization of the IL-4 receptor appears to be a critical first step in IL-4 mediated signal transduction.
One of the major physiologic roles of IL-4 is as a B lymphocyte activation and differentiation factor (Rabin, et al., 1985; Oliver, et al., 1985). The protein was first isolated based on this activity. In this regard, IL-4 activates production of IgG1 (Vitetta, et al., 1985), but is also responsible for isotype switching in B cells from production of IgG to IgE immunoglobulins (Coffman, et al., 1986; Lebman and Coffman, 1988, Del Prete, et al., 1988). The effect of IL-4 on the in vivo regulation of IgE has been clearly demonstrated. Neutralization of IL-4 by treatment with a monoclonal anti-IL-4 antibody (Finkelman, et al., 1986) or a monoclonal antibody to the IL-4 receptor (Finkelman, et al., 1990) will block the IgE response. A recombinant soluble IL-4 receptor has been shown to inhibit IgE production by up to 85% in vivo (Sato, et al., 1993). IL-4 deficient mice produced by gene-targeting in murine embryonic stem cells have normal B and T cell development, but serum levels of IgG1 and IgE are strongly reduced (Kuhn, et al., 1991). IL-4 augmented IgE production leads to an atopic state (allergy/asthma) (Finkelman, et al., 1989; Katona, et al., 1991).
The IL-4 mediated up-regulation of IgG1 may play a role in the inflammation cascade. IgG1 has recently been shown to form immune complexes which bind to the cellular receptors for the Fc domain of immunoglobulins (FcRs) leading to an inflammatory response (Sylvestre and Ravetch, 1994; Ravetch, 1994). IL-4 transgenic mice have been produced that hyperexpress IL-4 (Tepper, et al., 1990). These mice have elevated levels of serum IgG1 and IgE and they develop allergic inflammatory disease. These findings demonstrate the critical role IL-4 plays in the humoral immune response.
Another major physiologic role for IL-4 is as a T lymphocyte growth factor (Hu-Li, et al., 1987; Spits, et al., 1987). IL-4 enhances the proliferation of precursors of cytotoxic T cells (CTLs) and their differentiation into active CD8.sup.+ CTLs (Widmer and Grabstein, 1987; Trenn, 1988). IL-4 appears to augment the IL-2 driven induction of lymphokine-activated killer (LAK) cells (Higuchi, et al., 1989), which have been shown to lyse a variety of tumor cell targets without major histocompatibility complex (MHC) restriction. The role played by IL-4 at this crucial nexus of T cell effector function is fundamental to the success or failure of the immune response.
IL-4 has been shown to affect nonlymphoid hematopoietic cells in a variety of ways. IL-4 has been shown to modulate monocyte/macrophage growth (McInnes and Rennick, 1988; Jansen, et al., 1989) while enhancing their differentiation and cytotoxic activity for certain tumor cells (Crawford, et al., 1987; Te Velde, et al., 1988). IL-4 also has activity as a stimulant of mast cell growth (Mosmann, et al., 1986; Brown, et al., 1987), and increases production and recruitment of eosinophils (Tepper, et al., 1989).
IL-4 alone or in conjunction with other cytokines can promote the expression of a variety of cell-surface molecules on various cell types with diverse implications for disease. Specifically, IL-4 can interact with tumor necrosis factor (TNF) to selectively enhance vascular cell adhesion molecule-1 (VCAM-1) expression contributing to T cell extravasation at sites of inflammation (Briscoe, et al., 1992). IL-4 alone or in combination with TNF or IFN-gamma has been shown to increase both MHC antigen and tumor-associated antigen expression on a variety of neoplastic cells (Hoon, et al., 1991).
As detailed above, IgG1 immune complexes bind to the cellular receptors for the Fc domain of immunoglobulins (FcRs) leading to an inflammatory response. Inhibition of IL-4 and the subsequent reduction in IL-4 mediated IgG1 expression may prove efficacious against immune complex inflammatory disease states. Indeed, inhibitory ligands to IL-4 might also prevent the IL-4 mediated overexpression of VCAM-1, thus attenuating the ability of T cells to extravasate at sites of inflammation.
Inhibition of IL-4 activity with a monoclonal antibody, a recombinant soluble IL-4 receptor, or gene knock-out, results in a reduction of serum IgE levels. An inhibitory oligonucleotide ligand to IL-4 could be clinically effective against allergy and allergic asthma.
A recent report has described a disorder in bone homeostasis in transgenic mice that inappropriately express IL-4 under the direction of the lymphocyte-specific proximal promoter for the 1 ck gene (Lewis, et al., 1993). Bone disease in these mice resulted from markedly decreased bone formation by osteoblasts, features identical to those found in human osteoporosis. Inhibiting this IL-4 mediated reduction in osteoblast activity may prove beneficial against osteoporosis.
Graft-versus-host disease (GVHD) is a major complication of human tissue transplantation. GVHD does not exist as a single clinical manifestation but can involve immunologic abnormalities ranging from immunodeficiency to systemic autoimmunities (Ferrara, et al., 1991). These systemic autoimmunities include clinical and serological manifestations of human systemic lupus erythematosus (SLE). Several murine models of SLE have been developed (Gleichmann, et al., 1982; van Rappard-van Der Veen, et al., 1982), and the induction of systemic GVHD in mice has been described (Via, et al., 1988). Two recent studies have shown in vivo efficacy of a mouse monoclonal antibody to IL-4 in preventing GVHD and SLE in these murine model systems (Umland, et al., 1992; Ushiyama, et al., 1995). These observations suggest that an inhibitor of human IL-4 may be effective in treatment of chronic systemic autoimmunities such as SLE and GVHD.
A variety of microbicidal infections are characterized by depressed cellular but enhanced humoral immune responses, which suggests a T.sub.H2 type of response to infection. This T.sub.H2 phenotype is characterized by T cell secretion of IL-4, as detailed earlier. IL-4 blocks the microbicidal activity of IFN-gamma activated macrophages in fighting Leishmania major infection (Liew, et al., 1989; Leal, et al., 1993). Inhibition of IL-4 would enhance the T.sub.H1 effector arm of the immune response enhancing cellular immunity and leading to the resolution of infection. Neutralization of IL-4 in vivo allows mice otherwise susceptible to Leishmania major infection to fight off the parasite and clear the infection (Heinzel, et al., 1989). Several informative studies have looked at the T.sub.H1 /T.sub.H2 phenotypic distinction in infected mice, and suggest a T.sub.H1 dominated response being most effective in fighting microbial infection (for a review, see Sher and Coffman, 1992).
IL-10
IL-10 is a cytokine produced by the Th2 cells, but not Th1 cells, and inhibits synthesis of most of all cytokines produced by Th1 cells but not Th2 cells (Mosmann et al., 1991). In addition to the effect on CD4.sup.+ cells with Th1 phenotype, IL-10 also inhibits CD8.sup.+ T cells with "Th1-like" phenotype. IL-10 is a potent suppressor of macrophage activation. It can suppress the production of proinflammatory cytokines, including TNF.alpha., IL-1, IL-6, IL-8 and IFN-gamma. Overall, these results suggest that IL-10 is a potent macrophage deactivator and an effective anti-inflammatory reagent. In addition, IL-10 prevents the IFN-.gamma.-induced synthesis of nitric oxide, resulting in decreased resistance to intracellular parasites (Gazzinelli et al., 1992).
Both human and mouse (hIL-10 and mIL-10, respectively) have been cloned and expressed (Moore et al., 1990; Vieira et al., 1991). The two cDNAs exhibit high degree of nucleotide sequence homology (&gt;80%) throughout and encode very similar open reading frames (73% amino acid homology). Both proteins are expressed as noncovalent homodimers that are acid labile (Moore et al., 1993). Whether monomers are equally bioactive is not clear yet. Based on the primary structure IL-10 has been categorized into the four a-helix bundle family of cytokines (Shanafelt et al., 1991). Possibly due to high degree of sequence homology and similar structure hIL-10 has been shown to be active on mouse cells (Moore et al., 1993) but not vice a versa. hIL-10 is an 18 kDa polypeptide with no detectable carbohydrate; however, in mIL-10 there is one N-linked glycosylation. The recombinant hIL-10 has been expressed in CHO cells, COS7 cells, mouse myeloma cells, the baculovirus expression system and E. coli. The rIL-10 expressed in these systems have indistinguishable biological behavior (Moore et al., 1993).
Parasitic infection often leads to polarized immune response of either Th1 or Th2 type which can mediate protection or susceptibility. The outcome of a parasitic infection depends on the nature of the parasite and the host. The best understood example is Leishmania major infection in mice. L. major is a protozoan parasite that establishes an intracellular infection in macrophages, where it is mainly localized in phagolysosomes. Activated macrophages can efficiently destroy the intracellular parasite and thus parasitic protection is achieved by macrophage activation. Nonactivated macrophages do not kill these organisms. As expected, activation of macrophages upon IFN-gamma treatment enhanced the protection, whereas IL-4 and IL-10 blocked the increased microbicidal activity induced by IFN-gamma (Liew et al., 1989). In most inbred strains (example, C57/BL6) cutaneous infection of L. major often leads to localized infection with spontaneous healing and confers resistance to reinfection. However, in BALB/c mice, L. major infection induces nonprotective immune response by producing IL-4. The antibody response mediated by IL-4 is ineffective and leads to death (Howard et al., 1980). In healing strains a strong Th1 response has been noticed with high level of IFN-.gamma., whereas in susceptible BALB/c mice a nonproductive Th2 response with significant levels of IL-4 was found (Heinzel et al., 1991). Further it was shown that a single injection of monoclonal anti-IFN-gamma antibody can convert a resistance into a susceptible mouse (Belosevic et al., 1989). As expected, the treatment of BALB/c mice with anti-IL-4 antibody led to the development of Th1 response and healing (Sher & Coffman, 1992). Thus, depending on the nature of the pathogen, changing the immune response to a T cell subset with a protective phenotype can lead to therapeutic intervention of the disease state. Understanding the regulation between the Th1 and Th2 phenotype mediated by cytokines will help in designing cytokine-antagonist in therapeutics. The production of IL-10 is strongly increased in mice infected with various pathogens such as Leishmania major, Schistosoma mansoni, Trypanosoma cruzi and Mycobacterium Leprae (Sher, et al, 1992; Salgame et al., 1991, Heinzel et al., 1991).
When designing immune therapy to facilitate mounting the right arm of defense mechanism toward pathogens, it is important to maintain a balance between the two arms also. Th2-type responses may be important in controlling the tissue damage mediated by Th1 cells during the response to an intracellular infectious agent. Keeping some Th1 cells functioning in a predominantly Th2 environment can help abrogate damaging effects of Th1 by secreting IL-10 and IL-4. One extreme of the spectrum of Th1/Th2 is reflected in transgenic mice lacking the IL-10 gene (Kuhn et al., 1993). The IL-10 deficient mouse is normal with respect to its development of T and B cell subsets. However these mice develop chronic enterocolitis (or inflammatory bowel disease) due to chronic inflammation via continuous overproduction of cytokines such as TNF.alpha. and IFN-gamma(Th1 response).
IL-12 can also induce the development of the Th1 subset. By using Lysteria monocytogen, an intracellular gram-positive bacterium, infection in antibody T cell receptor transgenic mice as a model it has been shown that IL-10 can block the production of IL-12 from macrophages (Hsieh et al., 1993) Thus an IL-10-antagonist will tip the Th1/Th2 population predominantly to Th2 type environment by 1, preventing the inhibition of the production of Th1 cytokines 2 by allowing the production of a cytokine that induces the development of Th1 subset.
With experimental evidence in hand it has been proposed that the resistance and/or progression to AIDS is dependent on a Th1/Th2 stage of an individual (Clerici & Shearer, 1993). This hypothesis is based on the findings that progression to AIDS is characterized by loss of IL-2 and IFN-gamma production (loss of Th1 response) with increase in IL-4 and IL-10 (acquired Th2 response). Many seronegatives (HIV-exposed individuals) generate a strong Th1-type response. It is important to note that after seroconversion both IL-4 and IL-10 levels go up at the expense of IL-4 and IFN-gamma. However, in full-blown AIDS patients, Th2 response seems to be mediated by high levels of IL-10 but not with IL-4, the level of which goes down to normal in these individuals. An anti-IL-10 reagent may serve as a potential therapeutic in shifting the Th2 response to Th1 in AIDS patients to offer protection.
TNF.alpha.
TNF.alpha. is an extracellular cytokine and a central mediator of the immune and inflammatory response (Beutler et al., 1989; Vassalli, 1992). It is a homo-trimer (Smith et al., 1987, Eck et al., 1988), and has a subunit size of 17 kD. It circulates at concentrations of less than 5 pg/ml in healthy individuals (Dinarello et al., 1993) and it can go as high as 1000 pg/ml in patients with sepsis syndrome (Casey et al., 1993). The human TNF.alpha. is nonglycosylated, whereas in some other species (notably the mouse) glycosylation occurs on a single N-linked site in the mature protein, but the sugar moiety is not essential for biological activity (Beutler et al., 1989). The human TNF.alpha. is acidic with a pH of 5.3 (Aggarwal et al., 1985). Each TNF.alpha. subunit consists of an anti parallel .beta.-sandwich and it participates in a trimer formation by an edge-to-face packing of .beta.-sheets. The structure of the TNF.alpha. trimer resembles the "jelly-roll" structural motif characteristic of viral coat proteins (Jones et al., 1989). TNF.alpha. is a relatively stable molecule and may be exposed to chaotropic agents such as urea, SDS, or guanidinium hydrochloride, and renatured with recovery of as much as 50% of the initial biological activity. The TNF.alpha. renaturability may reflect the limited number of internal disulfide bonds (one per monomer) required for maintenance of structure (Beutler et al., 1989).
Another related molecule, TNF.beta., has the same bioactivity as TNF.alpha.. The interspecies sequence identity within the TNF.alpha. and TNF.beta. families is 71% and 61%, respectively (Beutler et al., 1989). The sequence identity between hTNF-.alpha. and hTNF-.beta. is only 29% (Beutler et al., 1989). Despite their low similarity, both hTNF.alpha. and hTNF.beta. bind to the same receptors with comparable affinities.
TNF.alpha. mediates its bioactivity through binding to cell surface receptors. The TNF.alpha. receptors are found on the surface of virtually all somatic cells tested (Vassalli, 1992). Two distinct TNF.alpha. receptors have been characterized of apparent molecular weights 55 kD (p55 TNF.alpha.-R1) and 75 kD (p75 TNF.alpha.-R2) (Hohmann et al., 1989; Brockhaus et al., 1990; Loetscher et al., 1991). Both receptors bind TNF.alpha. and TNF.beta. with high affinities (Kd=0.3-0.6 nM) (Loetscher et al., 1990; Schall et al., 1990; Pennica et al., 1992).
TNF.alpha. has diverse activities, and thus is implicated in several diseases as follows:
Septic shock. Sepsis incidents have been increasing for the last 60 years and is the most common cause of death in intensive care units in the United States (Parrillo, 1991). The mortality of septic shock remains at approximately 50% despite the standard use of aggressive antibiotics and cardiovascular support for the past 10 years (Parrillo, 1991). The evidence implicating TNF.alpha. in sepsis is as follows. Pretreatment of mice or baboons with monoclonal antibodies to TNF.alpha. protects them from lethal doses of E. coli LPS (Beutler et al., 1985). Anti-TNF.alpha. antibodies protect primates against lethal endotoxin sepsis and against lethal S. aureus-induced shock (Fiedler et al., 1992; Hinshaw et al., 1992). Soluble-TNF.alpha.-receptor (p55)-IgG-Fc fusions (TNF.alpha. receptor immunoadhesin) were found to protect mice from endotoxic shock, even when administered lhr after endotoxin infusion. The same immunoadhesin was also effective against listeriosis in mice (Haak-Frendscho et al., 1994). Another immunoadhesin based on the p75 receptor was also shown to be effective in lethal endotoxemia and it was functioning simultaneously as both TNF.alpha. carrier and TNF.alpha. antagonist (Mohler et al., 1993).
Cachexia. In vivo administration of TNF.alpha. causes cachexia in mice (Oliff et al., 1987). Therefore, TNF.alpha. antagonists may protect cancer or AIDS infected patients from cachexia.
Cerebral malaria. High levels of TNF.alpha. are associated with poor prognosis in children with cerebral malaria, and antibodies to TNF.alpha. protect mice from cerebral complications of Plasmodium berghei infection (Grau et al., 1987).
Arthritis. Antibodies to TNF.alpha. reduce the production of the inflammatory cytokine, IL-1 in synovial cells (Brennan et al., 1989). TNF.alpha. is an inducer of collagenase, the major destructive protease in rheumatoid arthritis (Brennan et al., 1989). Anti-TNF.alpha. antibodies were found to ameliorate joint disease in murine collagen-induced arthritis (Williams et al., 1992). Transgenic mice carrying the hTNF.alpha. gene develop arthritis which can be prevented by in vivo administration of a monoclonal antibody against hTNF.alpha. (Keffer et al., 1991).
Graft Rejection and Graft versus Host Reaction (GVHR). TNF.alpha. has been implicated in the acute phase of graft-versus-host disease and in renal allograft rejection. Antagonists of TNF.alpha. may then be able to prevent these life-threatening conditions. Anti-TNF.alpha. antibodies have been found to delay graft rejection in experimental animals (Piguet, 1992). Also, injection of anti-TNF.alpha. antibodies during the acute phase of GVHR reduces mortality, and the severity of intestinal, epidermal, and alveolar lesions (Piguet, 1992). Clinical trials of the efficacy of anti-TNF.alpha. antibody in human bone marrow transplantation are underway.
AIDS. Studies of intracellular signal transduction pathways revealed that TNF.alpha. induces proteins that bind to kB-like enhancer elements and thus takes part in the control of NF-kB-inducible genes (Lenardo et al., 1989; Lowenthal et al., 1989; Osborn et al., 1989). The antiviral activity of TNF.alpha. at least in part is mediated by the interaction of NF-kB with a virus-inducible element in the .beta.-interferon gene (Goldfeld et al., 1989; Visvanathan et al., 1989). By an analogous mechanism, TNF.alpha. appears to activate human immunodeficiency virus type I (Duh et al., 1989; Folks et al., 1989). Therefore, TNF.alpha. antagonists may prove useful in delaying the activation of the AIDS virus and may work in conjunction with other treatments in the cure of AIDS.
Parkinson's disease. Recently, elevated TNF.alpha. levels have been found in the brain and the cerebrospinal fluid of Parkinsonian patients (Mogi et al., 1994). This report speculates that elevated TNF.alpha. levels may be related to neuronal degeneration associated with the disease.
RANTES
RANTES is a small (MW 8-kD) highly basic (pI.about.9.5) chemokine that belongs to the CC group (Schall, 1991; Baggiolini et al., 1994). It does not appear to be glycosylated (Schall, 1991) and is a chemoattractant for monocytes (Schall et al., 1990; Wang et al., 1993; Wiedermann et al., 1993), basophils (Bischoff et al., 1993; Kuna et al., 1993), eosinophils (Rot et al., 1992), and CD4.sup.+ /UCHL1.sup.+ T lymphocytes which are thought to be prestimulated or primed helper T cells involved in memory T cell function (Schall et al., 1990). RANTES is not only a chemoattractant but it also stimulates cells to release their effectors leading to tissue damage. For example, RANTES causes histamine release from basophils (Kuna et al., 1992; Kuna et al., 1993; Alam et al., 1993). It also causes the secretion of eosinophil basic peptide (Alam et al., 1993) and the production of oxygen free radicals (Rot et al., 1992) by eosinophils.
Initially, it was thought that RANTES was synthesized by activated T cells but recently other cells were found to synthesize it very fast upon stimulation. RANTES mRNA is expressed late (3 to 5 days) after activation of resting T cells, whereas in fibroblasts, renal epithelial and mesangial cells, RANTES mRNA is quickly up-regulated by TNF.alpha. stimulation (Nelson et al., 1993).
Receptors for RANTES have been identified. There is a promiscuous receptor on the surface of erythrocytes that binds all chemokines with a Kd=5 nM (Horuk et al., 1993; Neote et al., 1993). This receptor is thought to be a sink for chemokines to help in the establishment of chemotactic gradients. Signal transducing receptors have also been identified and cloned (Gao et al., 1993; Neote et al., 1993; Van-Riper et al., 1993; Wang et al., 1993). Monocytes carry a G-protein coupled receptor that binds RANTES with estimated Kd of 400 pM, but also MCAF and MIP-1a with lower affinities (estimated Kd of 6 and 1.6 nM respectively) (Wang et al., 1993). A receptor molecule has been cloned from neutrophils that can bind RANTES with a lower affinity of about 50 nM (Gao et al., 1993).
Disease State. RANTES antagonists may have therapeutic application in inflammation. Blockage of the chemoattractant and effector cell activation properties of RANTES would block local inflammation and tissue damage. The mechanism of action of the RANTES antagonist will be the inhibition of RANTES binding to cell surface receptors.
RANTES is chemoattractant for monocytes, basophils, eosinophils and memory lymphocytes. Basophils are the major source of mediators such as histamine and peptido-leukotrienes, and are an essential element of the late-phase responses to allergens in hypersensitivity diseases. These cells are also involved in other inflammatory pathologies, including certain autoimmune reactions, parasitic infections and inflammatory bowel diseases. In these conditions, basophil recruitment and activation is independent of IgE. Numerous reports have accumulated over the years that describe the effects of a group of elusive stimuli operationally called "histamine-releasing factors." A large number of these elusive stimuli may well be contributed by RANTES.
Eosinophils also are important in allergic inflamation, and together with lymphocytes, form prominent infiltrates in the bronchial mucosa of patients with asthma. They are believed to be the cause of epithelial damage and the characteristic airway hyper-reactivity. The recruitment of lymphocytes of the Th2 type, which comigrate with eosinophils into sites of late-phase reactions, is an important source of other chemoattractant cytokines and growth factors that prime eosinophils.
RANTES, with its effects on monocytes, basophils, eosinophils and lymphocytes appears to be a potent stimulator of effector-cell accumulation and activation in chronic inflammatory diseases and in particular, allergic inflammation.
The recruitment system of inflammatory cells has some redundancy built into it. However, RANTES has some unique properties. It is a more potent chemoattractant than MCP-1 and MIP-1.alpha., while MCP-1 is more potent stimulator of histamine release from basophils (Baggiolini et al., 1994). RANTES causes the production of oxygen radicals by eosinophiles while MIP-1.alpha. cannot (Rot et al., 1992). RANTES is as potent as C5a in the recruitment of eosinphils, but not as potent a trigger of the eosinophil oxidation burst (Rot et al., 1992). C5a is a very potent chemoattractant: however, it lacks the specificity of RANTES. It attracts not only basophils and eosinophils but also neutrophils. Since the eosinophils, but not the neutrophils, are important in the pathophysiology of some inflammatory conditions, such as the allergen-induced late-phase reaction and asthma, specific chemoattractants such as RANTES are expected to be involved.
Using in situ hybridization, RANTES expression has been found in interstitial mononuclear cells and proximal tubular epithelial cells in human kidney transplants undergoing rejection. Antibody staining revealed the presence of RANTES not only within the interstitial infiltrate and renal tubular epithelial cells but also in high abundance in inflamed endothelium (Wiedermann et al., 1993). Based on these results a haptotactic mechanism was postulated. Haptotaxis is defined as cell migration induced by surface-bound gradients. The haptotactic mechanism was supported by in vitro experiments and anti-RANTES antibodies have been found to prevent that in vitro haptotaxis.
Human rheumatoid synovial fibroblasts express mRNA for RANTES and IL-8 after stimulation with TNF.alpha. and IL-1.beta. (Rathanaswami et al., 1993). There is a differential regulation of expression of IL-8 and RANTES mRNA. Cycloheximide enhanced the mRNA levels for IL-8 and RANTES after stimulation with IL-1.beta. but reduced the levels of RANTES mRNA after stimulation with TNF.alpha.. Also, IL-4 down-regulates and IFN-gamma enhances the TNF.alpha. and IL-1.beta. induced increase in RANTES mRNA, whereas the induction of IL-8 mRNA by TNF.alpha. or IL-1.beta. was inhibited by IFN-gamma and augmented by IL-4. Moreover, the combination of TNF.alpha. and IL-1.beta. synergistically increased the level of IL-8 mRNA, whereas under the same conditions, the levels of RANTES mRNA were less than those induced with TNF.alpha. alone. These studies suggest that the synovial fibroblasts may participate in the ongoing inflammatory process in rheumatoid arthritis, and RANTES might be one of the participating effectors. The observed differential regulation of IL-8 and RANTES indicates that the type of cellular infiltrate and the progress of the inflammatory disease is likely to depend on the relative levels of stimulatory and inhibitory cytokines.
RANTES has also been implicated in atherosclerosis and possibly in postangioplasty restenosis (Schall, 1991). The participation of MCP-1 in atherosclerosis has been studied to a greater extent. Recently mRNAs for RANTES, MIP-1.alpha. and MIP-1.beta. have been detected in in situ in normal carotid plaque and heart transplant atherosclerosis. RANTES mRNA is not detected in the same cells expressing MIP-1.alpha. and MIP-1.beta., but it is expressed in lymphocytes and macrophages typically more proximal to the lumen. The data argue for positive feed-back mechanisms for the CC chemokines and possible differential expression of these chemokines at various stages in the progression of arterial disease.
Finally, elevated RANTES levels have been correlated with endometriosis (Khorram et al., 1993). RANTES levels were elevated in pelvic fluids from women with endometriosis, and these levels correlate with the severity of the disease.
Protein Homology between Human and Animal. The murine RANTES has been cloned (Schall et al., 1992). Sequence analysis revealed 85% amino acid identity between the human and mouse proteins. The human and murine RANTES exhibit immune crossreactivity. Boyden chamber chemotaxis experiments reveal some lack of species specificity in monocyte chemoattractant potential, as recombinant muRANTES attracts human monocytes in a dose-dependent fashion in vitro. Also, hRANTES transfection into mouse tumor cell lines produce tumors in which the secretion of hRANTES by those tumors correlates with increased murine monocyte infiltration in vivo (Schall et al., 1992).
SELEX
A method for the in vitro evolution of nucleic acid molecules with highly specific binding to target molecules has been developed. This method, Systematic Evolution of Ligands by EXponential enrichment, termed SELEX, is described in U.S. patent application Ser. No. 07/536,428, entitled "Systematic Evolution of Ligands by Exponential Enrichment," now abandoned, U.S. patent application Ser. No. 07/714,131, filed Jun. 10, 1991, entitled "Nucleic Acid Ligands," now issued as U.S. Pat. No. 5,475,096, U.S. patent application Ser. No. 07/931,473, filed Aug. 17, 1992, entitled "Methods for Identifying Nucleic Acid Ligands," now U.S. Pat. No. 5,270,163 (see also WO91/19813), each of which is herein specifically incorporated by reference. Each of these applications, collectively referred to herein as the SELEX Patent Applications, describes a fundamentally novel method for making a nucleic acid ligand to any desired target molecule.
The SELEX method involves selection from a mixture of candidate oligonucleotides and step-wise iterations of binding, partitioning and amplification, using the same general selection scheme, to achieve virtually any desired criterion of binding affinity and selectivity. Starting from a mixture of nucleic acids, preferably comprising a segment of randomized sequence, the SELEX method includes steps of contacting the mixture with the target under conditions favorable for binding, partitioning unbound nucleic acids from those nucleic acids which have bound specifically to target molecules, dissociating the nucleic acid-target complexes, amplifying the nucleic acids dissociated from the nucleic acid-target complexes to yield a ligand-enriched mixture of nucleic acids, then reiterating the steps of binding, partitioning, dissociating and amplifying through as many cycles as desired to yield highly specific, high affinity nucleic acid ligands to the target molecule.
The basic SELEX method has been modified to achieve a number of specific objectives. For example, U.S. patent application Ser. No. 07/960,093, filed Oct. 14, 1992, entitled "Method for Selecting Nucleic Acids on the Basis of Structure, now abandoned (see U.S. Pat. No. 5,707,796" describes the use of SELEX in conjunction with gel electrophoresis to select nucleic acid molecules with specific structural characteristics, such as bent DNA. U.S. patent application Ser. No. 08/123,935, filed Sep. 17, 1993, entitled "Photoselection of Nucleic Acid Ligands, now abandoned," describes a SELEX based method for selecting nucleic acid ligands containing photoreactive groups capable of binding and/or photocrosslinking to and/or photoinactivating a target molecule. U.S. patent application Ser. No. 08/134,028, filed Oct. 7, 1993, entitled "High-Affinity Nucleic Acid Ligands That Discriminate Between Theophylline and Caffeine, now abandoned (see U.S. Pat. No. 5,580,737)," describes a method for identifying highly specific nucleic acid ligands able to discriminate between closely related molecules, termed Counter-SELEX. U.S. patent application Ser. No. 08/143,564, filed Oct. 25, 1993, entitled "Systematic Evolution of Ligands by EXponential Enrichment: Solution SELEX,", now abandoned (see U.S. Pat. No. 5,683,867) describes a SELEX-based method which achieves highly efficient partitioning between oligonucleotides having high and low affinity for a target molecule. U.S. patent application Ser. No. 07/964,624, filed Oct. 21, 1992, entitled "Nucleic Acids Ligands to HIV-RT and HIV-1 Rev" now issued as U.S. Pat. No. 5,496,938 describes methods for obtaining improved nucleic acid ligands after SELEX has been performed. U.S. patent application Ser. No. 08/400,440, filed Mar. 8, 1995, entitled "Systematic Evolution of Ligands by EXponential Enrichment: Chemi-SELEX,", now U.S. Pat. No. 5,705,337, describes methods for covalently linking a ligand to its target.
The SELEX method encompasses the identification of high-affinity nucleic acid ligands containing modified nucleotides conferring improved characteristics on the ligand, such as improved in vivo stability or improved delivery characteristics. Examples of such modifications include chemical substitutions at the ribose and/or phosphate and/or base positions. SELEX-identified nucleic acid ligands containing modified nucleotides are described in U.S. patent application Ser. No. 08/117,991, filed Sep. 8, 1993, entitled "High Affinity Nucleic Acid Ligands Containing Modified Nucleotides,", now abandoned (see U.S. Pat. No. 5,660,985) that describes oligonucleotides containing nucleotide derivatives chemically modified at the 5- and 2'-positions of pyrimidines. U.S. patent application Ser. No. 08/134,028, supra, describes highly specific nucleic acid ligands containing one or more nucleotides modified with 2'-amino (2'-NH.sub.2), 2'-fluoro (2'-F), and/or 2'-O-methyl (2'-OMe). U.S. patent application Ser. No. 08/264,029, filed Jun. 22, 1994, entitled "Novel Method of Preparation of Known and Novel 2' Modified Nucleosides by Intramolecular Nucleophilic Displacement," describes oligonucleotides containing various 2'-modified pyrimidines.
The SELEX method encompasses combining selected oligonucleotides with other selected oligonucleotides and non-oligonucleotide functional units as described in U.S. patent application Ser. No. 08/284,063, filed Aug. 2, 1994, entitled "Systematic Evolution of Ligands by Exponential Enrichment: Chimeric SELEX", now U.S. Pat. No. 5,647,459, and U.S. patent application Ser. No. 08/234,997, filed Apr. 28, 1994, entitled "Systematic Evolution of Ligands by Exponential Enrichment: Blended SELEX,", now U.S. Pat. No. 5,683,867, respectively. These applications allow the combination of the broad array of shapes and other properties, and the efficient amplification and replication properties, of oligonucleotides with the desirable properties of other molecules. Each of the above described patent applications which describe modifications of the basic SELEX procedure are specifically incorporated by reference herein in their entirety.